Hepadnavirus
The family hepadnaviridae is a family of enveloped animal viruses with a core of DNA that cause hepatitis B in humans. The hepadnaviridae are not responsible for human hepatitis A (a single-stranded RNA enterovirus), human hepatitis C (Flaviridae family of single stranded RNA virus), or human hepatitis D (a closed circular negative-sense RNA satellite virus, “delta virus”, that requires hepatitis B virus (HBV for replication). The hepadnaviridae family includes hepatitis viruses of other species, e.g. woodchuck, duck, ground squirrel, and heron, in addition to human and simian hepatitis B.
The HBV genome consists of a 3.2 kb partially double-stranded circular DNA with four overlapping reading frames that encode the viral DNA polymerase, the viral core antigen (HbcAg), the viral surface antigens and the X antigens (Seeger, C., and Mason, 2000, Microbiol Mol Biol Rev. 64 (1): 51-68; Tiollais et al., 1985, Nature 317 (6037): 489-495). There are four HBV transcripts synthesized: 3.5 kb, 2.4 kb, 2.1 kb and 0.7 kb. Synthesis of the HBV RNAs is under the control of the pregenomic/preC, S1, S2, X promoters and enhancer I, II (De Clercq, 1999, Int J Antimicrob Agents. 12(2): 81-95).
The 3.5 kb mRNA serves as the template for reverse transcription but also encodes the HBV DNA polymerase and the core antigen (Summers et al., 1982, Cell 29 (2): 403-415, Weimer et al., 1987, J Virol. 61 (10): 3109-3113).
Hepatitis B virus infection is a major health problem worldwide. Conservative estimates place the number of persons chronically infected with hepatitis B virus (HBV) at more than 300 million (Fu et al., 2000, Antimicrob Agents Chemother. 44: 3402-3407). This is a viral disease with a long incubation period (about 50 to 160 days) in contrast to Hepatitis A virus (infectious hepatitis virus) which has a short incubation period. The virus is usually transmitted by injection of infected blood or blood derivatives or merely by use of contaminated needles, lancets or other instruments. Clinically and pathologically, the disease is similar to viral hepatitis type A; however, there is no cross-protective immunity. HBV is a causative agent of both an acute and chronic form of hepatitis. More than 300 million people throughout the world are chronic carriers of HBV. Typically, the human host is unaware of infection and HBV infection leads to acute hepatitis and liver damage, abdominal pain, jaundice and elevated blood levels of certain enzymes. Additionally, HBV contributes to the formation of hepatocellular carcinoma and is second only to tobacco as a cause of human cancer. The mechanism by which HBV induces cancer is unknown, although it has been postulated that it may directly trigger tumor development or indirectly trigger tumor formation through chronic inflammation, cirrhosis and cell regeneration associated with the infection. Viral antigen (HBAg) is found in the serum after infection.
The best defense to date has been vaccination. Human serum-derived vaccines, through genetic engineering, have been developed. Although the vaccine has been found effective, production has been hampered by the limited supply of human serum from chronic carriers and a long and expensive purification process. Furthermore, each batch of vaccine must be tested in chimpanzees to ensure safety. Additionally, vaccines do not help the patients already infected with the virus.
Great efforts have been made to develop clinically useful treatments for hepatitis B but they have been met with limited success. For example, interferon and several nucleoside analogs have shown relatively low cure rate of hepatitis B and they have often produced serious adverse effects. IFN-α treatment has a very limited efficacy and patients have exhibited variable adverse effects (De Clercq, 1999, Int J Antimicrob Agents. 12(2): 81-95), which some patients cannot tolerate. Lamivudine (also known as 3TC) and adefovir are very potent HBV inhibition agents that target the HBV DNA polymerase (Chang et al., 1992. J. Biol. Chem. 267:13938-13942; Doong et al. 1991, Proc. Natl. Acad. Sci. U.S.A. 88: 8495-8499) and adefovir dipivoxil (Angus et al., 2003, Gastroenterology 125:292-297). 2′,3′-Dideoxycytidine (ddC) has been shown to have high toxicity on the central and peripheral nervous system. Another nucleoside analog, ara-AMP, was found to transiently suppress HBV infection but also has been shown to be extremely toxic as well.
Cyclopentyl purine derivatives have also shown anti-viral activity. The process for preparing such compositions have been disclosed in U.S. Pat. Nos. 4,999,428 and 5,015,739. Additionally, Onishi et al., in U.S. Pat. No. 5,777,116 have disclosed a method of making cyclopropane derivatives that include a xanthin-9-yl group.
Schinazi et al., in U.S. Pat. No. 5,684,010, have produced enationtiomerically pure beta-D-dioxolane nucleosides which show selective anti-hepatitis B activity. Additionally, Lin et al., in U.S. Pat. No. 5,830,881, have discovered that certain dideoxynucleoside analogs which contain a ribofuranosyl moiety having a L-configuration instead of the usual D-configuration showed potent inhibition of viral replication. However, unlike other nucleoside analogs, these analogs have shown very low toxicity to the host cells such as animal or human.
Hostetler, in U.S. Pat. No. 5,817,638, have produced nucleoside analogs such as 2′,3′-dideoxycytosine, which are linked through a 5′ phosphate of the pentose group to selected lipids such as dioleoylphosphatidylcholine. The lipophilic nature provides an advantage over the use of the nucleoside analog alone and makes it possible to incorporate them into the lamellar structure of liposomes. This form enables them to be taken up by liver cells which harbor the hepatitis B virus.
Processes have been disclosed by in U.S. Pat. Nos. 5,142,051, 5,641,763 and 5,869,467 for the production of N-(2-phosphonylmethoxyethyl) and N-(3-hydroxy-2-phosphonylmethoxypropyl) derivatives of pyrimidine and purine bases. These compounds also could include a xanthin-9-yl group. These compounds are regarded as acyclic analogues of nucleosides in which the nucleoside sugar moiety is replaced by a substituted carbon chain bearing hydroxy groups.
Although the compounds developed by Holly and others have not been tested specifically against hepatitis B viruses, they have shown in-vitro anti-viral activity against other DNA viruses such as the herpes viruses. Other analogs that show anti-Hepatitis B virus activity include phosphonomethyoxymethyl purine and pyrimidine derivatives as described by in U.S. Pat. Nos. 5,726,174 and 5,837,871.
Chang et al., on the other hand, in U.S. Pat. No. 5,929,038, have developed an anti-HBV compound that is an iridoid aglycone compound produced from the parent iridoid glycosides which are monoteropenoid compounds and are derived from medicinal plants. In addition to inhibiting HBV DNA synthesis, these compounds also protect the liver from hepatic damage such as that induced by carbon tetrachloride intoxication.
In the past, anti-HBV nucleotide analogues such as (−)-(2R,5S)-1-[2-(hydroxymethyl)oxathiolan-5-yl]cytosine (3TC), 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) and 9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]guanine (PCV) have been used in clinical trials. However, some HBV-infected patients often experience a recurrence of HBV after a period of treatment with 3TC or PCV; this recurrence is due to the emergence of viral resistance. Additionally, the 3TC-resistant HBV, for example, becomes cross-resistant to other anti-HBV nucleotide analogs.
Therefore, one major concern of nucleoside analogue treatment is the emergence of drug-resistant variants of HBV (Fu et al., 1999, Biochem Pharmacol. 57 (12):1351-1359; Leung et al., 2001, Hepatology. 33 (6): 1527-1532; Liaw et al., 2000, Gastroenterology. 119(1): 172-180). Given the longer period of treatment using lamivudine, resistance was shown to increase from 14% at 1 year to 57% and 70% for years 3 and 5 respectively, (Liaw et al., 2003, J Gastroenterol Hepatol. 18:239-245) with respect to patients treated with lamivudine. With the intensive efforts in the search for effective antiviral agents against drug-resistant HBV, some nucleotide analogues have been developed and are under clinical evaluation for the treatment of 3TC-resistant HBV infections (Mutimer, 2001, J. Clin. Virol. 21:239-242; Levine et al., 2002, Antimicrob. Agents Chemother. 46:2525-2532). Although adefovir is a new drug recently approved by the FDA, Angus et al. reported that a novel mutation in the HBV polymerase emerged, which rendered the HBV resistant to the antiviral treatment (Angus et al., 2003, Gastroenterology 125 (2):292-297).
RNAi and ribozymes have been used in down regulating HBV RNA and core protein expression (McCaffrey et al., 2003, Nature Biotechnol. 21 (6): 639-644: Shlomai et al., 2003, Hepatology. 37 (4): 764-770 and Morrisey et al., 2002, J Viral Hepat. 9 (6): 411-418). HBV core protein the major capsid protein of HBV. Its phosphorylated form might be important for pregenomic RNA packaging, and its native form is important for viral DNA replication (Lan et al., 1999, Virology. 259 (2): 342-348). Core protein also functions as a transcriptional activator of the HBV pregenomic/preC promoter (Kwon et al., 2002, Biochem Cell Biol. 80 (4):445-455). Butz et al. developed a peptide aptamer, which can bind to the core protein and interfere with capsid formation (Butz et al., 2001, Oncogene 20(45):6579-6586). Deres et al. (Deres et al., 2003, Science. 299 (5608): 893-896) discovered a non-nucleoside inhibitor of HBV nucleocapsid maturation. Both studies demonstrated that the inhibition of HBV replication could be achieved by interfering with the assembly of core protein and capsid maturation. McCaffrey and Shlomai also independently observed HBV DNA inhibition by siRNA directed to the core gene (McCaffrey et al., 2003, Nature Biotechnol. 21 (6):639-644; Shlomai et al., 2003, Hepatology. 37 (4):764-770). They targeted different sequences in the same core gene region and got different degrees of inhibition on HBV replication. However, establishing the RNAi, as a viable therapeutic approach requires resolving several major issues: persistence of the RNAi inhibitory effect, efficient delivery system, viral resistance (Gitlin et al., 2003, J. Virol. 77 (13):7159-7165) and stabilization of RNAi.
Flaviviruses
Flaviviruses belong to the genus Flavivirus of the family Togaviridae. According to virus taxonomy, about 50 viruses including Hepatitis C virus (HCV), Yellow Fever virus, Dengue Virus, Japanese Encephalitis virus, West Nile virus and related flaviviruses. The viruses belonging to the genus Flavivirus are simply called flaviviruses.
The flaviviruses are agents of infectious disease and predominate in East, Southeast and South Asia and Africa, although they may be found in other parts of the world as well. Japanese encephalitis virus is the causative agent of Japanese encephalitis (JE). The mortality rate from JE is rather high and the disease brings heavy sequelae. Although found in Japan, the disease has spread to other parts of Asia and is now found predominantly outside of Japan, primarily in South and Southeast Asia.
Yellow fever is a tropical mosquito-borne viral heptatitis, due to Yellow Fever virus (YFV), with an urban form transmitted by Aedes aegypti, and a rural, jungle or sylvatic form from tree-dwelling mammals by various mosquitoes of the Haemagogus species complex. Yellow fever is characterized clinically by fever, slow pulse, albuminuria, jaundice, and congestion of the face and hemorrhages, especially hematemesis (“black vomit”). It is fatal in about 5-10% of the cases.
Japanese encephalitis virus (“JEV”) is the causative agent of Japanese encephalitis (JE). JE is an epidemic encephalitis or encephalomyelitis of Japan, Russia (Siberia) and other parts of Asia. The mortality rate from JE is rather high and the disease brings heavy sequelae. Although found in Japan, the disease has spread to other parts of Asia and is now found predominantly outside of Japan, primarily in South and Southeast Asia.
West Nile virus is the causative agent of West Nile fever, a disease characterized by headache, fever, masculopapular rash, myalgia, lymphadenopathy and leukopenia. The virus is spread by Culex mosquitoes from a reservoir in birds.
Dengue is a disease of tropical and subtropical regions occurring epidemically and caused by Dengue virus, one of a group of arboviruses which causes the hemorrhagic fever syndrome. Four grades of severity are recognized: grade I: fever and constitutional symptoms, grade II: grade I plus spontaneous bleeding (of skin, gums or gastrointestinal tract), grade III: grade II plus agitation and circulatory failure and grade IV: profound shock. The disease is transmitted by a mosquito of the genus Aedes (generally A. aegyptiI, but frequently, A. albopictus). Also called Aden, bouquet, breakbone, dandy, date, dengue (hemorrhagic) or polka, solar fever, stiffneck fever, scarlatina rheumatica or exanthesis arthorosia. “Hemorrhagic dengue” is a more pathogenic epidemic form of dengue that has erupted in a number of epidemic outbreaks in the Pacific region in recent years.
Infection with dengue viruses is a major public health problem in tropical countries, especially in Southeast Asia and the Western Pacific, but dengue viruses may also be found in the Americas. As the dengue virus is transmitted to humans via the Aedes aegypti mosquito, it is not unexpected that the tropical and subtropical countries, in particular, those in Southeast Asia are highly endemic for dengue.
A major concern and increasing problem for public health officials has been the occurrence of severe complications which arise from dengue viral infections. Both dengue hemorrhagic fever (DBF) and shock syndrome (DSS) are clinical outcomes related to the presence of pre-existing immunity to a heterologous dengue virus serotype. Dengue hemorrhagic fever is initially characterized by a minor febrile illness lasting approximately 3-5 days. The patient may deteriorate at defervescence into the next phase of the syndrome with hemostatic disorders and increased vascular permeability frequently accompanied by internal bleeding and shock. As many as 1.5 million children are reported to have been hospitalized with 33,000 deaths from this syndrome since it was first recognized in Thailand in the 1950's. DHF/DSS has since continued to persist in South Asia. DHS/DSS is also found in a number of tropical or near tropical countries, including Cuba, Burma, Indonesia, India, Maldives, Sri Lanka and the South Pacific Islands. Dengue outbreaks are usually associated with a density of mosquito vectors, in particular, Aedes aegypti. 
Dengue viruses can be divided into 4 serotypes which are antigenically very similar to each other, but which differ enough to elicit only partial cross-protection after infection by one serotype. Such an infection by one serotype therefore, does not provide life-long immunity to the other serotypes. Vaccine approaches to preventing dengue infections have been unsuccessful to date.
The term “Hepatitis C Virus” or (HCV) is used throughout the specification to describe the hepatitis virus which is the causative agent of non-A, non-B hepatitis. The Disease in the acute stage is, in general, milder than hepatitis B, but a greater proportion of such infections become chronic. It is clinically diagnosed by a well-defined set of patient symptoms, including jaundice, hepatic tenderness and an increase in the serum levels of alanine aminotransferase and aspartate aminotransferase.
HCV is an enveloped virus containing a positive-sense single-stranded RNA genome of approximately 9.4 kb. The viral genome consists of a 5′ untranslated region (UTR), a long open reading frame encoding a polyprotein precursor of approximately 3011 amino acids, and a short 3′ UTR. The 5′ UTR is the most highly conserved part of the HCV genome and is important for the initiation and control of polyprotein translation. Translation of the HCV genome is initiated by a cap-independent mechanism known as internal ribosome entry. This mechanism involves the binding of ribosomes to an RNA sequence known as the internal ribosome entry site (IRES). An RNA pseudoknot structure has recently been determined to be an essential structural element of the UCV IRES. Viral structural proteins include a nucleocapsid core protein (C) and two envelope glycoproteins, E1 and E2. HCV also encodes two proteinases, a zinc-dependent metalloproteinase encoded by the NS2-NS3 region and a serine proteinase encoded in the NS3 region. These proteinases are required for cleavage of specific regions of the precursor polyprotein into mature peptides. The carboxyl half of nonstructural protein 5, NS5B, contains the RNA-dependent RNA polymerase. The function of the remaining nonstructural proteins, NS4A and NS4B, and that of NS5A (the amino-terminal half of nonstructural protein 5) remain unknown.
Colacino et al. in U.S. Pat. Nos. 5,821,242 and 5,891,874, have developed a series of benzimidazole compounds which inhibit replication in other flaviviruses such as Hepatitis C by interfering with the structure and function of the viral replication complex.
Ribavirin (1-beta-D-ribofuranosyl-1-1,2,4-triazole-3-carboxamide), which is structurally similar to guanosine, has in vitro activity against several DNA and RNA viruses including Flaviviridae (Davis, 2000, Gastroenterology 118:S104-S114). Ribavirin reduces serum amino transferase levels to normal in 40% or patients, but it does not lower serum levels of HCV-RNA (Davis, 2000, Gastroenterology 118:S104-S114). Thus, ribavirin alone is not effective in reducing viral RNA levels. Additionally, ribavirin has significant toxicity and is known to induce anemia.
Interferons (IFNs) are compounds that have been commercially available for the treatment of chronic hepatitis for nearly a decade. IFNs are glycoproteins produced by immune cells in response to viral infection. IFNs inhibit viral replication of many viruses, including HCV, and when used as the sole treatment for hepatitis C infection, IFN suppresses serum HCV-RNA to undetectable levels. Additionally, IFN normalizes serum amino transferase levels. Unfortunately, the effects of IFN are temporary and a sustained response occurs in only 8%-9% of patients chronically infected with HCV (Davis, 2000, Gastroenterology 118:S104-S114).
A number of patents disclose HCV treatments using interferon-based therapies. For example, U.S. Pat. No. 5,980,884 discloses methods for retreatment of patients afflicted with HCV using consensus interferon. U.S. Pat. No. 5,942,223 discloses an anti-HCV therapy using ovine or bovine interferon-tau. U.S. Pat. No. 5,928,636 discloses the combination therapy of interleukin-12 and interferon alpha for the treatment of infectious diseases including HCV. U.S. Pat. No. 5,908,621 discloses the use of polyethylene glycol modified interferon for the treatment of HCV. U.S. Pat. No. 5,849,696 discloses the use of thymosins, alone or in combination with interferon, for treating HCV. U.S. Pat. No. 5,830,455 discloses a combination HCV therapy employing interferon and a free radical scavenger. U.S. Pat. No. 5,738,845 discloses the use of human interferon tau proteins for treating HCV. Other interferon-based treatments for HCV are disclosed in U.S. Pat. Nos. 5,676,942, 5,372,808 and 5,849,696.
The U.S. FDA has approved Schering's Ribavarin product, Rebetol capsules to treat chronic HCV infection in combination with Schering's alpha interferon products. Hoffman La Roche is selling ribavirin under the name CoPegus, also for use in combination with interferon for the treatment of HCV.
Other approaches have been attempted and are reviewed by Bymock et al., in Antiviral Chemistry & Chemotherapy, 11:2; 79-95 (2000). A few of the more recent approaches attempted are reviewed below.
Specifically, a method for the treatment of hepatitis C infection (2004/0006007) and flaviviruses and pestiviruses) in humans and other host animals is disclosed that includes administering an effective amount of a biologically active 1′,2′, or 3′-branched beta-D or beta-L nucleosides or a pharmaceutically acceptable salt or prodrug thereof, administered either alone or in combination, optionally in a pharmaceutically acceptable carrier. WO 01/96353 discloses 3′-prodrugs of 2′-deoxy-beta-L-nucleosides for the treatment of HBV. U.S. Pat. No. 4,957,924 discloses various therapeutic esters of acyclovir. Other patent applications disclosing the use of certain nucleoside analogs to treat hepatitis C virus include: WO 01/32153, WO 01/60315, WO 02/057425, WO 02/057287, WO 02/18404. U.S. Pat. No. 6,323,180 and U.S. Pat. Pub. No. 2004/0033959 discloses the use of viral protease inhibitors to inhibit the replication of HCV.
Herpesviruses
Herpesviruses include Herpes Simplex Virus types 1 and 2 (HSV-1 and HSV-2), Human Cytomegalovirus (HCMV), Epstein-Barr Virus (EBV) and Equine herpesviruses 1 and 4 (EHV-1 and EHV-4). The herpesviruses comprise a large family of double stranded DNA viruses. They are also a source of the most common viral illnesses in man. Eight of the herpes viruses, herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (VZV), human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and human herpes viruses 6, 7, and 8 (HHV-6, HHV-7, and (HHV-8), have been shown to infect humans.
HSV-1 and HSV-2 cause herpetic lesions on the lips and genitals, respectively. They also occasionally cause infections of the eye and encephalitis. HCMV causes birth defects in infants and a variety of diseases in immune-compromised patients such as retinitis, pneumonia, and gastrointestinal disease. VZV is the causitive agent of chicken pox and shingles. EBV causes infectious mononucleosis and can also cause lymphomas in immune-compromised patients. It has been associated with Burkitt's lymphoma, nasopharyngeal carcinoma, and Hodgkins disease. HHV-6 is the causitive agent of roseola and may be associated with multiple sclerosis and chronic fatigue syndrome. HHV-7 disease association
However, not all patients are responsive and a large number fail this therapy. In fact, approximately 30-50% of patients ultimately fail combination therapy. Treatment failure in most cases is caused by the emergence of viral resistance. Viral resistance in turn is caused by the rapid turnover of HIV-1 during the course of infection combined with a high viral mutation rate. Under these circumstances incomplete viral suppression caused by insufficient drug potency, poor compliance to the complicated drug regimen as well as intrinsic pharmacological barriers to exposure provides fertile ground for resistance to emerge.
Arylnaphthalene Lignan Lactones
Arylnaphthalene lignan lactones are natural products found in plant species and many of them exhibit diverse biological activities, such as phosphodiesterase inhibition, (Ukita et al., 1999, J Med. Chem. 42:1293-1305) leukotriene biosynthesis inhibition, (Thérien et al., 1993, Bioorg. Med. Chem. Lett. 3:2063-2066) and hypolipidemic, (Iwasaki et al., 1995, Chem. Pharm. Bull. 43:1701-1705) antitumoral (Ward et al., 1997, Nat. Prod. Rep. 14:43-74) and antiviral activities (Cow et al., 2000, Can. J. Chem. 78: 553-561, Charlton et al., 1998, J. Nat. Prod. 61:1447-1451). Helioxanthin is an arylnaphthalene lignan lactone isolated from the root of Heliopsis scabra Dunal (Compositae) (Burden et al., 1968, Tetrahedron Lett. 1035-1039) and the whole plant of Taiwania cryptomerioides Hayata (Taxodiaceae), (He et al., 1997, J. Nat. Prod. 60: 38-40) and the synthesis of which have been carried out by the inter- or intramolecular Diels-Alder reactions (Holmes et al., 1971, J. Chem. Soc. (C) 2091-2094; Stevenson et al., 1989, J. Nat. Prod. 52:367-375; Charlton et al., 1996, J. Org. Chem. 61:3452-3457) and benzannulation reaction (Mizufune et al., 2001, Tetrahedron Lett. 42:439).
U.S. Pat. No. 6,306,899 discloses that helioxanthin and certain analogues decreased the RNA level of HBV and antigen expression as well as selectively inhibited HBV replication in the cell culture model. This class of compound offers unique characteristic in anti-HBV chemotherapy. Therefore, it is of interest to synthesize more analogues of helioxanthin for studying structure-activity relationships and developing more selective, potent antiviral agents.